Associate Professor North Carolina State University
Recombineering is a homologous recombination-based method of gene modification that takes advantage of recombinant phage proteins expressed in E. coli to introduce insertions, deletions, substitutions and rearrangements in bacterial or eukaryotic genes of interest. To modify plant genes, recombineering is usually performed in engineered strains of E. coli in the context of transformable bacterial artificial chromosomes (BACs) harboring large (20-150Kb) plant genomic DNA fragments. Once edited, these modified BACs can be trimmed (also via recombineering) to remove the undesired portions of genomic DNA flanking the edited gene of interest and re-introduced back into plants via standard Agrobacterium-mediated transformation. Our group developed a series of optimized protocols and versatile genetic tools for efficient plant gene recombineering at increased throughput. We have built a series of tagging and trimming cassettes and dedicated vectors that enable the construction of additional cassettes and the transfer of modified genes from BACs to transformation-competent vectors via recombineering. The utility of the new tools is illustrated by the tagging of over 200 hormone-related genes and the characterization of the expression patterns of the 14 genes involved in the indole-3-pyruvate-dependent route of auxin biosynthesis in Arabidopsis.